1. Field of the Invention
This invention relates generally to compositions of and methods for obtaining cellulose synthase. The invention relates as well to the DNA sequences encoding cellulose synthase, the recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors and recombinant cellulose synthase polypeptides. More specifically, the invention relates to the cloning and expression of cellulose synthase from Acetobacter xylinum. 
2. Description of the Related Art
Cellulose biosynthesis is an event largely associated with plant cells, especially certain agronomic species such as cotton where as much as 90-95% of the secondary wall of mature cotton fibers is composed of cellulose. However, studies of cellulose biosynthesis in higher plants have been very frustrating, mainly because cellulose synthase, due to its lability, has evaded isolation and purification. Furthermore, the study of this enzyme has been hampered by the great difficulty in detecting formation of cellulose in vitro using purified preparations of plant and other eukaryotic cells [Delmer 1987: In higher plant preparations, the xcex2-1,3-glucan (callose) chains are synthesized instead of the xcex2-1,4-glucan of cellulose].
Apart from the higher plants, a large number of bacteria synthesize cellulose, including Acetobacter xylinum (A. xylinum), which converts as much as 35% of the glucose supplied to cultures of this bacterium into cellulose. Unlike the situation in plants and other eukaryotic cells noted above, the cellulose synthase of this bacterium has been isolated to a certain degree. Because of these facts, coupled with the capacity of the bacterium to synthesize a highly purified form of cellulose in vitro using membrane preparations, A. xylinum has become a preferred system for studies on the synthesis and organization of cellulose [Delmer 1987: In the bacterium, xcex2-1,4-glucan (cellulose) chains can be detected].
The biosynthesis of cellulose in A. xylinum is visualized as a two-step process. The first step involves the polymerization of sugar nucleotides (UDP-glucose; UDP-glc) into a xcex2-1,4,-linked glucan chain. The polymerization reaction is catalyzed by the enzyme cellulose synthase (UDP-glucose:1,4-xcex2-D-glucosyltransferase; E.C. 2.4.1.12) which is present in the cytoplasmic membrane (Bureau and Brown 1987). The activity of this enzyme is regulated by bis-(3xe2x80x2, 5xe2x80x2)-cyclic diguanylic acid (Ross 1987).
Purification of the cellulose synthase activity from membrane preparations has been accomplished by an entrapment procedure (Lin and Brown 1989) similar in some aspects to one used in the purification of chitin synthase from yeast (Kang 1984). The molecular weight of the native enzyme in Triton X-100 solubilized preparations appears to be 490 kd as determined by gel filtration (Lin and Brown 1989). Electron microscopy of the purified preparation shows doughnut-shaped particles indicating that the cellulose synthase may be organized as a tetramer or octamer (Lin and Brown 1989). Lithium dodecyl sulfate polyacrylamide gel electrophoresis of the purified preparation shows two major bands with molecular weights of 93 kd and 83 kd. The resistance of the cellulose synthase activity and of the 83 kd polypeptide to trypsin treatment has suggested that the 83 kd polypeptide is the active cellulose synthase (Lin and Brown 1989).
Photoaffinity probes have been used with glucan synthases to identify these enzymes in a variety of different species. In red beets, a 57 kd polypeptide has been shown to be the substrate-binding component of (1,3)-xcex2-glucan synthase (Wasserman 1989). In cotton fibers and in mung bean, a 50 kd polypeptide appears to bind the substrate (Delmer and Solomon 1989). In A. xylinum, Mayer et al. (1989) suggested a 67 k polypeptide as the substrate-binding subunit and a 57 kd polypeptide as the activator-binding subunit of a 420 kd oligomeric cellulose synthase. These investigators suggested the presence of similarly sized peptides in other cellulose-producing organisms such as Agrobacterium tumefaciens, mung bean, wheat, pea and cotton on the basis of immunochemical analyses.
Genes involved in the synthesis of other exopolysaccharides in a number of bacteria have been cloned using the standard approach of genetic complementation [Easson (1987); Harding (1987); Calvin and Hanawalt (1988)]. Mutants of A. xylinum defective in the production of cellulose, yet which still possess a normal complement of cellulose synthase have been identified in particular strains. Certain of these mutants have been shown to be deficient in the activity of UDPG-pyrophosphorylase, the enzyme required for the synthesis of the cellulose synthase substrate molecule, UDP-glucose. Complementation of these mutants by cloned fragments from A. xylinum-derived DNA has led to the isolation of the gene encoding UDPG-pyrophosphorylase (Valla 1989).
Surprisingly, however, no mutants actually deficient in cellulose synthase activity have been identified. This is despite the fact that there have been a large number of mutants identified which morphologically appear to be cellulose-deficient, but which synthesize small amounts of an altered crystalline polymorph of cellulose (cellulose II) and have the wild type level of cellulose synthase activity when assayed in vitro for cellulose synthesis [Saxena and Brown (1989); Roberts et al. (1989)].
Thus, it has been considerably difficult to apply the standard approaches of classical genetics or even the powerful techniques of molecular biology to the study of cellulose biosynthesis. Lacking purified quantities of the enzyme, lacking detailed information of the subunit architecture, lacking means to specifically identify the substrate-binding subunit, and lacking a simple genetic method of isolating mutants deficient in cellulose synthase activity, have each hampered the ability of researchers to isolate DNA segments encoding cellulose synthase.
The present invention for the first time, provides the vectors, DNA segments, purified protein, antibodies, methods of cloning, and recombinant host cells, seeds and plants necessary to obtain and use a recombinant cellulose synthase. Thus, the difficulties encountered with applying the standard approaches of classical genetics or techniques of molecular biology to the study of cellulose biosynthesis have been overcome. Accordingly, the present invention concerns generally compositions and methods for the preparation of recombinant cellulose synthase of both prokaryotic and eukaryotic origin.
In certain general and overall embodiments, the invention concerns recombinant vectors and isolated DNA segments encoding a cellulose synthase peptide. The DNA segments of the invention may encode biologically functional equivalent protein or peptides which have variant amino acid sequences, such as with changes selected based on considerations such as the relative hydropathic score of the amino acids being exchanged.
In the context of the present invention, the term cellulose synthase is intended to refer to peptides or proteins having the biological and the immunological identity of the cellulose synthase of the cell enabled lines by the present invention. For example, such cell lines would include cells of Acetobacter xylinum. Generally, the cellulose synthase of the invention will refer to a 723 amino acid peptide or protein (SEQ ID NO:2) in that this is the precise length of the only presently sequenced cellulose synthase. However, the invention does not preclude and, in fact enables, preparation or use of shorter or longer peptides or proteins, so long as a peptide or protein has similar in kind biological activity and/or a cross reactive immunological reactivity, for example, as defined by rabbit polyclonal antisera. For instance, the other wild type strain of A. xylinum ATCC 23769 which was used by the present inventors possesses a 75 kD polypeptide as a catalytic subunit of cellulose synthase by using the methods of the invention.
In certain general aspects, the invention relates to the preparation and use of DNA segments, including vectors or DNA fragments, having a sequence encoding a cellulose synthase polypeptide. For vectors, any number are known in which DNA sequences of the invention may be incorporated. The vector pUC18 has been demonstrated to be of particular value. Likewise, the related vectors M13mp18 and M13mp19 may be used in certain embodiments of the invention, in particular, in performing dideoxy sequencing.
In certain embodiments, the vector will contain a substantially purified DNA fragment which encodes at least a useful portion of a cellulose synthase polypeptide which includes the amino acids 1 to 723 of FIG. 1A-FIG. 1L, (SEQ ID NO:2) functionally equivalent amino acids. Recombinant vectors and isolated segments may, therefore, variously include the basic cellulose synthase coding region itself or may contain coding regions bearing selected alterations or modifications in the basic coding region of cellulose synthase. Alternatively, such vectors or fragments may encode larger proteins or peptides which nevertheless include the basic coding region. In any event, it should be appreciated that due to codon redundancy, as well as biological functional equivalence, this aspect of the invention is not limited to the particular DNA sequences shown in FIG. 1A-FIG. 1L, (SEQ ID NO:1).
Recombinant vectors such as the foregoing are useful both as a means for preparing quantities of the cellulose synthase-encoding DNA itself, and as a means for preparing the encoded protein and peptides. It is contemplated that where cellulose synthase proteins of the invention are made by recombinant means, one may employ either prokaryotic or eukaryotic expression and shuttle systems.
Prokaryotic host cells are disclosed in a preferred embodiment of the invention. However, in that prokaryotic systems are usually incapable of correctly processing eukaryotic precursor proteins, and since eukaryotic cellulose synthases are anticipated using the teachings of the disclosed invention, one may desire to express such sequences in eukaryotic hosts. However, even where the DNA segment encodes a eukaryotic cellulose synthase, it is contemplated that prokaryotic expression will have some additional applicability. Therefore, the invention can be used in combination with vectors which can shuttle between the eukaryotic and prokaryotic cells. Such a system is that of the Ti plasmids used in conjunction with the bacteria Agrobacterium tumefaciens. 
Where expression of cellulose synthase in a eukaryotic host is contemplated, it most likely will be desirable to employ a vector such as a plasmid, that incorporates a eukaryotic origin of replication, such as those of the CaMV (cauliflower mosaic virus) and plasmids derived therefrom. Additionally, for the purposes of expression in eukaryotic systems, one will desire to position the cellulose synthase encoding sequence adjacent to and under the control of an effective eukaryotic promoter such as promoters used in combination with Ti plasmids. To bring a coding sequence under control of a promoter, whether it is eukaryotic or prokaryotic, what is generally needed is to position the 5xe2x80x2 end of the translation initiation site of the proper translational reading frame of the protein between about 1 and about 50 nucleotides 3xe2x80x2 of or xe2x80x9cdownstreamxe2x80x9d with respect to the promoter chosen. Furthermore, where eukaryotic expression is anticipated, one will typically desire to incorporate into the transcriptional unit which includes the cellulose synthase, an appropriate polyadenylation site. Typically, the polyadenylation site is placed about 30-2000 nucleotides xe2x80x9cdownstreamxe2x80x9d of the termination site of the protein at a position prior to transcription termination.
Accordingly, in certain preferred embodiments, the vectors of the invention are those where the cellulose synthase polypeptide encoding sequence is positioned adjacent to and under the control of an effective promoter. The vectors may be that set of vectors known well to those of skill in the art where the promoter comprises a prokaryotic promoter, the vector being adapted for expression in a prokaryotic host. Alternatively, the vectors may be those of common knowledge to skilled artisans where the promoter comprises a eukaryotic promoter, and the vector further includes a polyadenylation signal positioned 3xe2x80x2 of the carboxy-terminal amino acid, and within a transcriptional unit of the encoded protein.
In certain embodiments of the invention, it is contemplated that DNA fragments both shorter and longer which incorporate sequences from FIG. 1A-FIG. 1L, (SEQ ID NO:1) will find additional utilities, including uses as short DNA fragment hybridization probes, e.g., in screening both prokaryotic and eukaryotic recombinant clone banks. In any.event fragm ents, corresponding to the sequence in FIG. 1A-FIG. 1L, (SEQ ID NO:1) stretches as short as 14 or so nucleotides, will generally find utility in accordance with these or other embodiments. By having stretches of at least about 14 nucleotides in common with the cellulose synthase DNA sequence of FIG. 1A-FIG. 1L, (SEQ ID NO:1) or it complement, a DNA segment will typically have the ability to form a preferential hybridization with cellulose synthase species DNA, particularly under more stringent conditions such as 0.15 M sodium chloride and 0.02 M sodium citrate, pH 7.4 at about 50xc2x0 C. While such a complementary or common stretch will typically ensure the ability to form a stable hybrid, longer stretches of complementary DNA may prove more desirable for certain embodiments. Thus, one may desire to use DNA segments incorporating longer stretches of complementarity, for example, on the order of 18, 22 or even 25 or so bases.
The invention also provides methods for isolating cellulose synthase polypeptides from both recombinant and non-recombinant sources. Such a protein will typically include an amino acid sequence corresponding to amino acids 1 to 723 of FIG. 1A-FIG. 1L, (SEQ ID NO:2). However, them methods, of the invention have been demonstrated to be successful in isolating a 75 kd cellulose synthase polypeptide from A. xylinum strain ATCC 23769. It will be obvious to those of skill in the art that this 75 kd polypeptide is different in molecular weight from the 83 kd polypeptide described extensively herein. Furthermore, it will be obvious to such skilled artisans that the differences in molecular weights of these two polypeptides may result from actual differences in the primary structure of the amino acid chains themselves or may result from any number of post-translational modifications. Since such a protein represents the sequence for the catalytic subunit of the cellulose synthase protein, such a protein may be used directly to synthesize cellulose when provided the proper environment and substrate. Additionally, such a protein may be used to prepare an antibody for use in certain embodiments which antibody may be either a polyclonal or a monoclonal antibody and which, in any case, represents an antibody immunologically reactive with any of the polypeptides of the invention.
The invention provides, therefore, a method of producing a recombinant cellulose synthase polypeptide. This method includes the use of a recombinant host cell where the recombinant host cell is capable of expressing a recombinant cellulose synthase polypeptide. Furthermore, the method for producing recombinant cellulose synthase provided in the invention includes culturing the host cell under conditions appropriate for expressing the polypeptide. Finally, the method of production claimed would include collecting the polypeptide thus expressed.
The method is particularly applicable where one desires to obtain a polypeptide corresponding to an 83 kd catalytic subunit of cellulose synthase of Acetobacter xylinum. However, it is proposed that the method may be directed to isolation of a cellulose synthase polypeptide and a gene that is encoding a protein that is substantially similar to the 83 kd catalytic subunit.
In particular embodiments, the method will typically involve selecting cells that are capable of cellulose synthesis, particularly cells of Acetobacter xylinum. A variety of cells are amenable to the method of the invention, for instance, cells of Agrobacterium, Rhizobium, Alcaligenes and particularly Sarcina. Of course, methods will typically involve culturing cells such that cellulose synthase is produced. In most cases, the cell lines used in the method will be one of those cell lines which can actively engage in cellulose biosynthesis without addition of extraneous activators. Where cells of Acetober xylinum will be used, cultures will be grown in aerated and agitated culture medium containing Celluclast in order to free the cells trapped in the product cellulose so that greater numbers of cells can be produced as an inoculum (see U.S. patent application, Ser. No. 022,904, filed Mar. 6, 1987, Brown et al.).
In certain embodiments, cellulose synthase may be partially purified from cells by solubilizing the cellular membranes. Partially purifying cellulose synthase from cells by solubilizing the cellular membranes is preferably accomplished using digitonin. However, other solubilizing agents may be employed as long as the cellulose synthase enzyme thus solubilized retains a substantial amount of its native activity. Other solubilizing agents which may be used include n-octyl glucoside and other nonionic detergents or Triton X-100.
Moreover, one may desire to even further purify the enzyme. If so, it has been found that a particularly useful approach employs xe2x80x9cproduct entrapmentxe2x80x9d. Product entrapment is explained in greater detail in the examples which follow. This technique entails generally layering solubilized enzyme solution on top of a cushion, e.g., of buffered glycerol, allowing synthesis of cellulose to occur, centrifuging the resulting mixture and recovering the supernatant. This may even be followed by a series of chromatographic steps to still further purify the protein.
After obtaining a partially purified cellulose synthase one may desire to admix it with a cellulose synthase activator in order to enhance the activity of the purified cellulose synthase. A useful cellulose synthase activator is bis-(3xe2x80x2,5xe2x80x2)-cyclic diguanylic acid, this molecule being the only such activator currently identified.
The present inventors have found a photoincorporation method to function in identifying the active subunit of the cellulose synthase. Photoincorporation may be desirable where other modes of labelling the active enzyme are insufficient. Photoincorporation of cellulose synthase is accomplished most readily using a radioactively labeled azidonucleotide analog capable of specifically interacting with the nucleotide binding site of cellulose synthase.
Upon sequencing the purified cellulose synthase one may construct oligonucleotide probes corresponding generally to some portion of the DNA segment which would encode such amino acid sequences as are determined from amino acid sequencing the purified cellulose synthase. Oligonucleotide probes which incorporate particular DNA sequences will preferably correspond to at least a portion of the amino acid sequences or various cellulose synthase polypeptides in accordance herewith, and may be synthesized by a variety of methods. Preferably, these probes will be DNA sequences which encode portions of the Acetobacter xylinum 83 kd cellulose synthase polypeptide. In any case, the resulting probes will be used to probe a suitable source of DNA from a cell line capable of cellulose synthesis.
The probing will usually be accomplished by hybridizing the oligonucleotide probes to a DNA source suspected of possessing a cellulose synthase gene. In some cases, the probes will constitute only a single probe, in others, the probes will constitute a collection of probes based on a certain amino acid sequence of the cellulose synthase and will account in their diversity for the redundancy inherent in the genetic code.
A suitable source of DNA from a cell line capable of cellulose synthase expression may be a genomic library of the cell line of interest. Alternatively, the suitable source of DNA may include total DNA from the cell line of interest. Once the hybridization process of the invention has identified a candidate DNA segment, one will desire to confirm that a positive clone has been obtained, e.g., by further hybridization, sequencing and/or expression and testing.
The invention also provides a means for obtaining a variety of recombinant host cells which incorporate a DNA, sequence in accordance with that depicted in FIG. 1A through is (SEQ ID NO:2). The host cell may be either prokaryotic or eukaryotic in nature. In any case, it is understood that the DNA segment encoding a cellulose synthase polypeptide will also possess the regulatory signals functional in the particular host cell. A preferred embodiment includes a recombinant plant entity which may comprise a plant cell (e.g., tissue cultured plant cells), a recombinant seed, or a recombinant plant having an incorporated gene encoding for a cellulose synthase polypeptide. The plant entity will possess the recombinant gene as a result of the in vitro introduction of the gene into a plant cell, wherein the recombinant gene is under the transcriptional control of regulatory signals functional in the particular plant entity. These regulatory signals will be appropriately selected to control the expression of the recombinant cellulose synthase in a manner to allow all the requisite transcriptional and post-transcriptional modification.
Of particular interest in this regard will be plant entities which are cultivated for their content of cellulose and, most particularly, will be such plant entities as species of the genus Gossypium. Alternatively, more primitive entities such as algae or cyanobacterium are included within the description. Importantly, the gene encoding cellulose synthase may either be a gene which is heterologous to the plant cell, seed or plant in which the gene is introduced or it may be a copy of the gene homologous to that found in the plant entity being transformed. As used herein, the terms heterologous and homologous refer to the source of the recombinant DNA in reference to the DNA of the host cell. Thus, A. xylinum DNA from a particular strain of this bacterium which is encoding a cellulose synthase polypeptide if used to transform another strain of cellulose synthase-producing host cell bacteria or if used to transform a eukaryotic host cell would be the incorporation into either host cell of a DNA segment heterologous to that of the host cell""s DNA. Conversely, the incorporation of the same A. xylinum DNA as above by retransforming an identical strain of A. xylinum would be the incorporation of a DNA segment homologous to that of the host cell""s DNA.
In certain general aspects, then, a method of producing a cellulose synthase polypeptide is provided by the invention. First, one produces a recombinant host cell according to the methods and with the compositions of the invention such that the recombinant cell so produced is capable of expressing the polypeptide. Next, one cultures the host cell under conditions appropriate for expressing the polypeptide. Finally, according to the methods and with the compositions of the invention the recombinant polypeptide is recovered.
In other general aspects, a method of producing a xcex2-1,4 glucan polymer is provided using the methods and compositions of the invention. First, one obtains one of the recombinant host cells of the invention using the methods and compositions of the invention such that the host cell so obtained is capable of producing the xcex2-1,4 glucan polymer. Next, one cultures the host cell under conditions appropriate for producing the xcex2-1,4 glucan polymer. Finally, one recovers the xcex2-1,4 glucan polymer thus produced.
It will be obvious to those of skill in the art that the recombinant cellulose synthase polypeptide expressed by these host cells may use as its sole substrate UDP-glucose and produce the homoploymer cellulose. Alternatively, the substrate for the recombinant polypeptide may be other nucleotide or mixtures of nucleotides with UDP-glucose such as UDP-xylose and will result it heteropolymers. Furthermore, the recombinant cellulose synthase may act alone in these host cells or may act in concert with other enzymes to produce mixed polymer compositions.